I. Angiogenesis
Angiogenesis, also called neovascularization, involves the formation of sprouts from preexistent blood vessels and their invasion into surrounding tissue. During angiogenesis, vascular endothelial cells re-enter the cell cycle, degrade underlying basement membrane, and migrate to form new capillary sprouts. These cells then differentiate, and mature vessels are formed. This process of growth and differentiation is regulated by a balance of pro-angiogenic and anti-angiogenic factors. A related process, vasculogenesis, involves the differentiation of endothelial cells and angioblasts that are already present throughout a tissue, and their subsequent linking together to form blood vessels.
Angiogenesis occurs extensively during development, and also occurs in the healthy body during wound healing in order to restore blood flow to tissues after injury or insult. Angiogenesis, however, has also been implicated in the development of certain diseases, including cancer and tumor formation. Indeed, the quantity of blood vessels in a tumor tissue is a strong negative prognostic indicator in breast cancer (Weidner et al., J. Natl. Cancer Inst. 84:1875-1887, 1992), prostate cancer (Weidner et al., Am. J. Pathol. 143:401-409, 1993), brain tumors (Li et al., Lancet 344:82-86, 1994), and melanoma (Foss et al., Cancer Res. 56:2900-2903, 1996). Angiogenesis has also recently been implicated in other disease states in many areas of medicine, including rheumatology, dermatology, cardiology and ophthalmology. In particular, undesirable or pathological tissue-specific angiogenesis has been associated with certain specific disease states including, for example, rheumatoid arthritis, atherosclerosis, psoriasis, diabetic retinopathy, and macular degeneration. (See, e.g., Fan et al., Trends Pharmacol. Sci. 16:57, 1995; Folkman, Nature Med. 1:27, 1995.) Furthermore, the alteration of vascular permeability is thought to play a role in both normal and pathological physiological processes (Cullinan-Bove et al., Endocrinol. 133:829, 1993; Senger et al., Cancer and Metastasis Reviews 12:303, 1993). Although the angiogenic process in each of these diseases is likely to share many features with developmental angiogenesis and tumor angiogenesis, each may also have unique aspects conferred by the influence of surrounding cells.
Multiple molecular mediators of angiogenesis have been identified including basic and acidic fibroblast growth factors (aFGF, bFGF), transforming growth factors alpha and beta (TGFα, TGFβ), platelet-derived growth factor (PDGF), angiogenin, platelet-derived endothelial cell growth factor (PD-ECGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF). Other stimulators implicated in angiogenesis include angiopoietin-1, Del-1, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), leptin, midkine, placental growth factor, pleiotrophin (PTN), progranulin, proliferin, and tumor necrosis factor-alpha (TNF-alpha). In addition, control of angiogenesis is further mediated by a number of negative regulators of angiogenesis produced by the body including angioarrestin, angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, gro-beta, heparinases, heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12, kringle 5 (plasminogen fragment), metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor-4 (PF4), prolactin 16 kD fragment, proliferin-related protein (PRP), retinoids, tetrahydrocortisol-S, thrombospondin-1 (TSP-1), vasculostatin, and vasostatin (calreticulin fragment).
Among these angiogenic regulators, VEGF appears to play a key role as a positive regulator of the abnormal angiogenesis accompanying tumor growth (reviewed in Brown et al., Control of Angiogenesis (Goldberg and Rosen, eds., 1996); Birkhauser et al., J. Biol. Chem. 271:603-606, 1996). Furthermore, recently the role of the PDGF family of signaling molecules has been under investigation, since it appears to play a role in the formation, expansion and proper function of perivascular cells, sometimes referred to as mural cells, e.g., vascular smooth muscle, mesangial cells, and pericytes.
II. VEGF-A
VEGF-A (polynucleotide and polypeptide sequences shown in SEQ ID NOs: 1 and 2, respectively) is a secreted, disulfide-linked homodimeric glycoprotein belonging to the VEGF/PDGF (platelet-derived growth factor) group of the cystine-knot superfamily of hormones and extracellular signaling molecules (see Vitt et al., Mol. Endocrinol., 15:681-694, 2001), which are all characterized by the presence of eight conserved cysteine residues forming the typical cystine-knot structure (named after cystine, a dimer of two cysteines linked by a disulfide bond). Five human VEGF-A isoforms of 121, 145, 165, 189 or 206 amino acids in length (VEGF-A121-206), encoded by distinct mRNA splice variants, have been described, all of which are capable of stimulating mitogenesis in endothelial cells. These isoforms differ in biological activity, receptor specificity, and affinity for cell surface- and extracellular matrix-associated heparan-sulfate proteoglycans, which behave as low affinity receptors for VEGF-A: VEGF-A121 does not bind to either heparin or heparan-sulfate; VEGF-A145 and VEGF-A165 (GenBank Acc. No. M32977) are both capable of binding to heparin; and VEGF-A189 and VEGF-A206 show the strongest affinity for heparin and heparan-sulfates. VEGF-A121, VEGF-A145, and VEGF-A165 are secreted in a soluble form, although most of VEGF-A165 is confined to cell surface and extracellular matrix proteoglycans, whereas VEGF-A189 and VEGF-A206 remain associated with extracellular matrix. Both VEGF-A189 and VEGF-A206 can be released by treatment with heparin or heparinase, indicating that these isoforms are bound to extracellular matrix via proteoglycans. Cell-bound VEGF-A189 can also be cleaved by proteases such as plasmin, resulting in release of an active soluble VEGF-A110.
Most tissues that express VEGF-A are observed to express several VEGF-A isoforms simultaneously, although VEGF-A121 and VEGF-A165 are the predominant forms, whereas VEGF-A206 is rarely detected (see Ferrara, J. Mol. Med. 77:527-543, 1999). VEGF-A145 differs in that it is primarily expressed in cells derived from reproductive organs (see Neufeld et al., FASEB J. 13:9-22, 1999). Human VEGF-A165, the most abundant and biologically active form, is glycosylated at Asn74 and is typically expressed as a 46 kDa homodimer of 23 kDa subunits.
Four cell-surface receptors that interact with VEGF-A have been identified. These include VEGFR-1/Flt-1 (fins-like tyrosine kinase-1; GenBank Acc. No. X51602; De Vries et al., Science 255:989-991, 1992); VEGFR-2/KDR/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-1; GenBank Acc. Nos. X59397 (Flk-1) and L04947 (KDR); Terman et al., Biochem. Biophys. Res. Comm. 187:1579-1586, 1992; Matthews et al., Proc. Natl. Acad. Sci. USA 88:9026-9030, 1991); neuropilin-1 (Gen Bank Acc. No. NM003873), and neuropilin-2 (Gen Bank Acc. No. NM003872). VEGF121 and VEGF165 bind VEGFR-1; VEGF121, VEGF145, and VEGF165 bind VEGFR-2; VEGF165 binds neuropilin-1; and VEGF165 and VEGF145 bind neuropilin-2. See, e.g., Neufeld et al., FASEB J. 13:9-22, 1999; Stacker and Achen, Growth Factors 17:1-11, 1999; Ortega et al., Fron. Biosci. 4:141-152, 1999; Zachary, Intl. J. Biochem. Cell Bio. 30:1169-1174, 1998; Petrova et al., Exp. Cell Res. 253:117-130, 1999.
VEGF-A-driven angiogenesis has a major role in the pathogenesis of diverse human diseases, including cancer, eye disorders, and rheumatoid arthritis. See Carmeliet et al., Nature 407:249-257, 2000. Recognition of the importance of VEGF-A for the development of several important classes of cancer recently culminated in the approval of AVASTIN™, a humanized monoclonal antibody to VEGF-A, for the treatment of metastatic colorectal cancer. See Ferrara et al., Nat. Rev. Drug Discov. 2004, 3:391-400, 2004. Similarly, the importance of VEGF-A in the pathogenesis of neovascular ocular disorders is reflected in the recent approval of LUCENTIS™, a humanized monoclonal antibody fragment, for the treatment of neovascular (wet) age-related macular degeneration (AMD).
III. PDGFRβ
PDGFRβ (platelet-derived growth factor receptor β; polynucleotide and polypeptide sequences shown in SEQ ID NOs. 3 and 4, respectively) is one of two structurally related cell surface receptor tyrosine kinases (PDGFRα and PDGFRβ) mediating the biological activities of various platelet-derived growth factor (PDGF) isoforms—PDGF-A, -B, -C, and -D. PDGFs belong to the PDGF/VEGF (vascular endothelial growth factor) family, which, as previously indicated, is characterized by the presence of eight conserved cysteine residues forming the typical cystine-knot structure. Two forms of the PDGF-A chain, containing 196 and 211 amino acid residues resulting from differential splicing of the transcript, are synthesized, dimerized, proteolytically processed in the N-terminus, and secreted from the cell as a ˜30 kDa dimer (Bonthron et al., Proc. Natl. Acad. Sci. USA 85:1492-1496, 1988; Rorsman et al., Mol. Cell. Biol. 8:571-577, 1988). The PDGF-B chain encoding 241 amino acid residues is dimerized, processed by additional proteolysis, and secreted as a 24 kDa dimer (Ostman et al., J. Biol. Chem. 263:16202-16208, 1988; Ostman et al., J. Cell. Biol. 118:509-519, 1992). The A and B chains are capable of forming both homodimers and heterodimers with each other (PDGF-AA, -BB, and -AB). The full-length PDGF-C and -D proteins contain 345 and 370 amino acid residues respectively, and both have a unique two-domain structure with a N-terminal CUB domain and a C-terminal PDGF/VEGF domain. Proforms of PDGF C and D are secreted as an approximately 85 kDa homodimer after cleavage of the N-terminal 22 signal peptide residues. Whereas secreted PDGF-AA, -BB, and -AB can readily activate their cell surface receptors, proteolytic removal of the CUB domain is required for the growth factor domain of PDGF-CC and -DD homodimers to activate the cell surface receptors
Both PDGFRs (PDGFRα and PDGFRβ) contain five extracellular immunoglobulin-like domains, a transmembrane domain, a juxtamembrane domain, splitted kinase domains, a kinase insert domain, and a cytoplasmic tail. These two receptors share 31% identity in the ligand binding domain, 27% identity in the kinase insert and 28% identity in the C-terminus, whereas they are 85% and 75% identical in the two halves of the kinase insert domain (Matsui et al., 1989; Rosenkranz and Kazlauskas, 1999). The three dimeric PDGF receptors (αα, αβ, ββ) mediate PDGF isoform-specific signal transduction. PDGF-AA effectively activates only PDGFRαα, PDGF-AB can activate either PDGFRαα or PDGFRαβ, while PDGF-BB activates all three dimeric PDGF receptors (see Claesson-Welsh et al., Mol. Cell. Biol. 8:3476-3486, 1988; Matsui et al., Science 243:800-804, 1989; Claesson-Welsh, J. Biol. Chem. 269, 32023-32026, 1994). The growth factor domain of PDGF-CC activates both PDGFRαα and PDGFRαβ, and the growth factor domain of PDGF-DD activates PDGFRββ and PDGFRαβ (see Li et al., Nature Cell. Biol. 2:302-309, 2000; Bergsten et al., Nature Cell. Biol. 3:512-516, 2001; Gilbertson et al., J. Biol. Chem. 276:27406-27414, 2001; LaRochelle et al., Nat. Cell. Biol. 3:517-521, 2001).
PDGFs produced by endothelial cells in vessels promote recruitment and proliferation of vascular smooth muscle cells/pericyte progenitors expressing PDGFR (Betsholtz et al., 2001). Chemotactic and mitogenic activities mediated by the PDGF/PDGFR paracrine signaling loop are crucial for the formation, branching and maintenance of blood vessels. As in embryogenesis, PDGF plays a critical role for angiogenesis in human tumors. Tumor angiogenesis, required for tumor outgrowth and metastasis, is a complex and highly regulated process involving many different cell types and extracellular factors. Endothelial cells and smooth muscle cells are the major components of blood vessels, and VEGF/PDGF super family members are among the critical mediators of tumor angiogenesis. Clinical studies revealed a correlation between vascular counts and expression frequency of VEGF and PDGF in tumors (Anan et al., Surgery 119:333-339, 1996). PDGFs directly and indirectly stimulate the angiogenic processes. PDGF released by the tumor cells induce migration of endothelial cells and vascular smooth muscle cells (vSMC), and also stimulate proliferation of these cells, suggesting a direct role of PDGFs in angiogenesis (Thommen et al., J. Cell. Biochem. 64:403-413, 1997). PDGFs were shown to induce transcription and secretion of VEGF-A by PDGFRβ expressing endothelial cells, suggesting an indirect role for PDGF induced angiogenesis (Wang et al., Cancer Res. 59:1464-1472, 1999). PDGFs also mediate the paracrine signaling loop between endothelial cells and vSMC/pericytes during tumor angiogenic processes. While PDGF-BB, -AB, and the growth factor domain of PDGF-CC induce indistinguishable angiogenic responses in mouse cornea assay, PDGF-AA (which binds to PDGFRαα but not PDGFRαβ or PDGFRββ) stimulates only a weak response (Cao et al., FASEB J. 16:1575-1583, 2002). This suggests that PDGFRα and PDGFRβ may differently regulate angiogenic processes, and points to the PDGFRβ subunit in particular as an important mediator of PDGF-induced angiogenesis.
PDGF receptor signaling has been linked to various processes in the disease states described above, including autocrine growth factor signaling in tumor cells, tumor and ocular angiogenesis and recruitment of regulation of stromal cells, namely fibroblasts in the tumor, or ocular disease tissues. Expression of almost all ligands of the PDGF family in NIH3T3 cells leads to transformation of the cell to a cancerous phenotype (Reviewed in Ostman and Heldin, Adv in Can Res 97:247-74, 2007). In support of this, co-expression of various PDGF ligands and receptors have been demonstrated in multiple diseases, including various cancers (reviewed in Ostman Cytokine and Growth Factor Rev 15:275-86, 2004). Furthermore, mutational activation of PDGF or PDGF receptors have now been shown to be associated with different malignancies, including dermatofibrosarcoma protuberans (DFSP), gastrointestinal stromal tumors (GIST) and Bcr-Abl-negative chronic myeloid leukemias (reviewed in Ostman and Heldin, Adv. Cancer Res. 97:247-74, 2007). The PDGF family has also been shown to play a significant role in tumor angiogenesis, especially with respect to recruitment of pericytes and vascular smooth muscle cells to the tumor and ocular vasculature. These mural cells (pericytes and smooth muscle cells) are thought to provide a supportive framework for growing vascular endothelial cells. PDGFRβ has been shown to be essential for the recruitment of pericytes to tumor vasculature and for the differentiation of mesenchymal stem cells to pericytes (Song et al., Nat. Cell Biol. 7:870-79, 2005; Bergers et al., Neuro. Oncol. 7:452-64, 2005). PDGFR antagonists have shown to inhibit angiogenesis by not only inhibiting pericyte recruitment but also by reducing endothelial cell coverage within tumors (Bergers et al., J. Clin. Invest. 111:1287-95, 2003). Furthermore, PDGF and PDGF receptors are expressed significantly in tumor stroma, namely fibroblast cells within various cancers and multiple experiments have demonstrated a critical role for PDGF-BB and PDGFRβ and PDGFRα receptors in stromal cell recruitment within tumors (reviewed in Ostman and Heldin, Adv. Cancer Res. 97:247-74, 2007). A series of recent studies indicate a function of these receptors in controlling tumor transvascular transport. Multiple pieces of evidence now support a role for PDGF and PDGFR in controlling interstitial fluid pressure (IFP), a key parameter determining transvascular transport. Most solid tumors are characterized by high IFP leading to decreased convection rate across capillary walls and there reduced uptake of drugs (chemotherapy) by tumors. PDGF receptor antagonists have shown to reduce tumor IFP and thereby allowing for increased drug uptake within tumors, leading to better anti-tumor efficacy (Pietras et al., Cancer Res. 62:5476-84, 2002; Pietras et al., Cancer Res. 61:2929-34, 2001). PDGFR antagonists therefore provide a method to inhibit multiple processes within the tumor vasculature, including autocrine effects on tumor cells, angiogenesis and affecting IFP mediated by tumor stroma. Combinations with other drugs, namely anti-angiogenec inhibitors like VEGF antagonists and/or chemotherapy may provide additional benefit to cancer patients.
IV. Inhibition of VEGF and PDGF Pathways
Although anti-angiogenic therapies, including AVASTIN™, have been approved for various cancers (and LUCENTIS™ for AMD), efficacy is moderate and patients eventually progress. One factor limiting efficacy is the presence of other angiogenic pathways that are not inhibited by these therapies. In mouse models, it has recently been demonstrated that PDGFRβ-expressing pericytes are found in tumors treated with VEGF antagonists and these provide a framework for newly formed endothelial cells to grow within the tumors (Mancuso et al., J. Clin. Invest. 116:2610-21, 2006) Inhibiting both the VEGF and PDGFR pathways may provide additive or synergistic angiogenesis inhibition in disease settings and, in cancer, may provide for enhanced delivery of chemotherapeutics by normalizing vessels and IFP.
Scientific evidence supports this therapeutic approach. Data that shows co-targeting both PDGFR and VEGFR signaling more effectively inhibits growth of endothelial vessels and is more effective at inhibiting tumor growth in preclinical disease models. In a spontaneous pancreatic tumor model (RIP-Tag), Bergers and colleagues demonstrated that combination with a VEGF inhibitor (SU5416) and a PDGF inhibitor (SU6668 or imatinib) inhibited growth of pancreatic adenocarcinomas when administered late during tumor progression (IT or RT), whereas VEGF inhibition alone was only effective when used in early disease settings (PT and IT) (Bergers et al., J. Clin. Invest. 111:1287-95, 2003). The decrease in tumor growth was associated with a decrease in tumor associated endothelial cells and pericytes and inhibition of angiogenesis. Similarly, in a pancreatic xenograft model (BxPC-3), treatment with both anti-PDGFRβ and VEGFR2 antibodies showed significant anti-tumor effects when compared to monotherapy treatment regimens (Shen et al., Biochem Biophys Res Commun 357:1142-47, 2007). Pietras and collegues further demonstrated that efficacy was substantially higher in the Rip-Tag pancreatic model when anti-VEGF and anti-PDGF therapies were combined together with chemotherapy (Pietras and Hanahan, J. Clin. Oncol. 23:939-52, 2005). Combination efficacy has also been demonstrated in ovarian carcinoma xenograft models combing PDGFR and VEGFR inhibitors (Lu et al., Clin. Cancer Res. 13:4209-17, 2007). These data provide strong proof-of-concept rationale for targeting both pathways in oncology. Furthermore, combination targeting of these pathways also has shown to inhibit neovascularization in a rodent eye model (Jo et al., Am. J. Pathol. 168:2036-53, 2006), providing preclinical support for possible efficacy in ocular disease indications, including AMD.